Surveillance of endemic coronaviruses during the COVID‐19 pandemic in Iran, 2021–2022

Abstract Background Human coronaviruses (HCoVs) 229E, OC43, HKU1, and NL63 are common viruses that continuously circulate in the human population. Previous studies showed the circulation of HCoVs during the cold months in Iran. We studied the circulation of HCoVs during coronavirus disease 2019 (COVID‐19) pandemic to find the impact of pandemic on the circulation of these viruses. Methods As a cross‐sectional survey conducted during 2021 to 2022, of all throat swabs sent to Iran National Influenza Center from patients with severe acute respiratory infection, 590 samples were selected to test for HCoVs using one‐step real‐time RT‐PCR. Results Overall, 28 out of 590 (4.7%) tested samples were found to be positive for at least one HCoVs. HCoV‐OC43 was the most common (14/590 or 2.4%), followed by HCoV‐HKU1 (12/590 or 2%) and HCoV‐229E (4/590 or 0.6%), while HCoV‐NL63 was not detected. HCoVs were detected in patients of all ages and throughout the study period with peaks in the cold months of the year. Conclusions Our multicenter survey provides insight into the low circulation of HCoVs during the COVID‐19 pandemic in Iran in 2021/2022. Hygiene habits and social distancing measures might have important role in decreasing of HCoVs transmission. We believe that surveillance studies are needed to track the pattern of HCoVs distributions and detect changes in the epidemiology of such viruses to set out strategies in order to timely control the future outbreaks of HCoVs throughout the nation.


| INTRODUCTION
Human coronaviruses (HCoVs) are pathogenic viruses of humans with a high zoonotic potential. 1 These viruses are believed to be originated and evolved in animals and known to be characterized by a crown-like appearance and a large, nonsegmented, positive, single-stranded ribonucleic acid. 2 There are seven strains of HCoVs infectious to humans recognized as HCoV-OC43, HCoV-NL63, HCoV-HKU1, HCoV-229E, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. 3 Like coronaviruses of mammalian, HCoVs are classified into alpha-CoVs and beta-CoVs, while the infectious viruses for birds mostly belong to the genera of gamma-CoVs and delta-CoVs. 4 Among infectious strains for humans, HCoV-229E and HCoV-NL63 have a bat origin and are alpha-CoVs, while HCoV-OC43 and HCoV-HKU1 are beta-CoVs known to be hosted by rodents. 2 HCoVs are important infectious agents in the etiology of both upper and lower respiratory tract infections (URTIs and LRTIs). 5 URTIs are often caused by HCoV-229E and HCoV-OC43 while LRTIs are mainly linked to HCoV-HKU1 and HCoV-NL63 5. All these four HCoVs are low pathogenic and endemic in humans. 6,7 Besides these viruses, three CoVs, namely, SARS-CoV, MERS-CoV, and SARS-CoV-2, are highly infectious for humans, which are associated with serious healthrelated problems and death. 8 While seasonal viruses are widely distributed worldwide, their detection rate generally relies on the time and location they are active. In the current situation when SARS-CoV-2, the most recent and newly emerged beta-coronavirus, is still challenging in almost all countries of the world such as Iran with millions of affected lives since early 2020, most seasonal viruses especially those targeting the human respiratory system (e.g., respiratory syncytial virus [RSV] and influenza viruses), which were active in the years just before the emergence of SARS-CoV-2, have mysteriously disappeared. 9 This dramatic reduction or even delayed in typical seasons of viral circulation that is being reported by surveillance data from different countries of the world is believed to be attributable to a series of nonpharmaceutical-mandated interventions (NPIs) (i.e., travel restrictions, borders and school closure, and hand and respiratory hygiene) used to limit the virus spread during the pandemic. 10 In Iran, a broad range of local restrictions along with recommended measures (i.e., wearing facial masks and washing hands) for being protected against COVID-19 have been introduced to residents immediately after the official announcement of the pandemic in early 2020. 11 To the best of our knowledge, this study is the first national

| Patients and specimen collection
To determine the impact of the pandemic on the circulation of seasonal HCoVs through 2021/2022, we designed a cross-sectional study that was conducted during the period of October 2021 through and October 2022. About 590 throat swabs were randomly selected from respiratory samples, which were routinely screened through the national influenza surveillance by the National Influenza Center (NIC), which is located at the Virology Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. These samples were obtained according to the Ministry of Health (MOH) instructions from those with SARI, who visited the medical care centers for at least one of the following symptoms: High fever (>38 C) and cough with onset within the last 10 days, which requires hospitalization. All the studied samples were collected in 2-3 mL of viral transport medium, refrigerated at or below 4 C, and sent within 48 h together with a standardized test request sheet, which included patient details (e.g., age, sex, and the place of residence) and medical records (e.g., symptoms and potential risk factors such as comorbidities) from different clinics across the country (e.g., Tehran, Alborz, Arak, Ardabil, East Azerbaijan, West Azerbaijan, Bushehr, Isfahan, Fars, Golestan, Kerman, Lorestan, Razavi Khorasan, Qom, Semnan, Zanjan, and Yazd).
The samples were aliquoted into two microtubes (one microtube for influenza/SARS-CoV-2 testing and one microtube for HCoVs) and stored at À70 C to maintain their quality intact until tested. Written informed consent was obtained from adults and the parents/ guardians of included children/infants before sampling by MOH.

| Viral genome extraction and detection
The inclusion criteria of sample selection for HCoVs testing were negative for influenza and SARS-CoV-2 infections. The SARS-CoV-2 RNA extraction kit (Viragene, Tehran, Iran) was used for ribonucleic acid isolation from the respiratory samples based on the manufacturer's protocol. After extraction, the total nucleic acids were assessed for concentration and purity by NanoDrop spectrophotometer (Thermo Fisher Scientific, USA) and a final volume of 50 μL of eluted ribonucleic acid stored in RNase free micro-tubes at À70 C for further analysis. All samples were tested for each of the four seasonal HCoVs.
SuperScript III Platinum One-Step qRT-PCR Kit (Invitrogen, USA) was

| Statistical analysis
Statistical analyses were performed using the R software (R version 4.2.1; 2022-06-23 ucrt). Pearson's chi-squared and Fisher's exact tests were used for comparisons in terms of categorical variables. A value of P < 0.05 was defined as the statistical level of significance.

| Demographics, prevalence, and seasonality
For over 12 months of surveillance, a total of 590 patients who complained of SARI with no confirmed influenza and SARS-CoV-2 infections were tested for four endemic HCoVs. As represented in Table 1 HCoVs, 16 (57%) in males, and 12 (43%) in females. However, the results showed no significant association between gender and positivity for HCoV infection (P > 0.05) ( Table 2). In our analysis, 17 (61%) and 11 (39%) were with and without severe illness based on their requirement for hospital admission, respectively (  (Tables 4 and 5). Interestingly, HCoV-NL63 was not detected. In our survey, cases infected with HCoVs were residents throughout the country as follows:   for his symptoms which were sore throat, dry cough, and chills.
The disease has also been represented as a matter of concern in countries in the middle east region including Iran with 7.5 million infected and thousands of deaths since early 2020 (https://www.  We showed that men were more likely to be infected with HCoV-OC43 than women, a finding that is consistent with the study T A B L E 3 Comparison of severity of illness, sex differences, and clinical manifestations in HCoV-OC43 infected and noninfected individuals. by E. R. Gaunt et al. 16 We also found that more males than females were infected with HCoV-229E. However, in the mentioned study, equal numbers of males and females were susceptible to infection with HCoV-229E. In a study from Iran, the majority of cases with HCoV-229E were male, suggesting likely a sex preference for this viral infection. 17 In the case of HCoV-HKU1, both our patients and patients of other studies had relatively same number of men and women with no statistically sex preference. 16 HCoVs can be detected year-round; however, in agreement with the results of recent epidemiological studies, our analysis showed that seasonal HCoVs are more common to be detected in cold months of the winter suggesting a strong seasonal distribution for all endemic HCoVs. 18   runny nose, cough, and shortness of breath and for HCoV-NL63 fever, cough, rhinorrhea, tachypnea, hypoxia, and obstructive laryngitis (croup). 3 Most of these symptoms were observed in our studied subjects regardless of the infective strain of the detected HCoVs. In our HCoVs-infected individuals, fever, cough, and rhinorrhea were among the most common symptoms similar to the findings of other studies. 5 As part of this surveillance, we investigated HCoV-OC43 infection. This viral infection is generally not specified to a specific season as this virus infection was diagnosed throughout the year of 2021/2022. However, its rate of detection peaked in December and January based on our results and the results of other studies. 22 HCoV-OC43 was also the most predominant HCoVs in other countries like Belgium, especially in children and the elderly. 15  HCoV-HKU1 was reported to be the cause of 0.04% to 2.1% of adult respiratory diseases around the world, 25 and also in Iran based on our analysis, which was detected in 2% of studied subjects with a respiratory infection. HCoV-HKU1 was found as both a single and mixed with other respiratory viruses in studies from Iran. 26 In our investigation, similar to other studies, respiratory symptoms were the T A B L E 5 Comparison of severity of illness, sex differences, and clinical manifestations in HCoV-229E infected and noninfected individuals. most common clinical manifestations of HCoV-HKU1; however, nonrespiratory involvements are also described such as gastrointestinal symptoms (e.g., diarrhea). 25 Most of the HCoV-HKU1-infected cases in our study (10 out of 12 virus-infected cases) were hospitalized for the severity of their illness. In agreement with this finding, there is some evidence regarding the requirement of oxygen and intensive care beds for critical care of patients with HCoV-HKU1 infection showing that a general belief that HKU1 causes only mild respiratory infections are not true all the time. 25 HCoV-HKU1-caused severe respiratory diseases, however, might be rare, and it can occur in immunocompromised and/or even sometimes in immunocompetent individuals. 27,28 Our virus-infected cases had no history of underlying diseases to discuss.
HCoV-NL63 was first detected in Iran in 2012 in a newborn girl who had suffered from respiratory distress. 29 Since the first detection, the virus infection has been diagnosed in a few studies across the nation. 23,26 An investigation from Iran (before the pandemic) in a similar duration time as our study found HCoV-NL63 by using the same method as our method in nasal and throat swabs of 23.9% of cases (specified children aged <5 years) who had acute respiratory infections. 23 Given the differences between the inclusion criteria of this study compared with ours, different results could be explained. In addition to this study, there were some pieces of evidence from different countries with a lower detection rate (for instance, 1.2% in Japan, 30 2.1% in Australia, 31 and 2.5% in Canada 32 ), regarding the virus infection. Here, we found no HCoV-NL63 in any of the samples during the study period (even in winter season when the virus is in highest detection rate in many countries 33 ) contrary to what has been detected in the years just before the beginning of the pandemic in Iran 23 and other parts of the world. 5 Of note, this finding is not surprising because other national surveillance before the pandemic with smaller sample sizes than ours 17 or by different methods of detection as our used method 34 found no evidence of HCoV-NL63 in studied cases with respiratory infections. HCoV-NL63 is more common in children; however, in our study, none of the children was diagnosed with this virus infection, which is consistent with reports outside Iran. 33 Given the fact that changes in the epidemiological pattern of common respiratory viruses had been observed and have already been described even in recent months in the course of the COVID-19 pandemic, 12  HCoV-HKU1) in their included subjects. 26 A report from Thailand showed young children with acute LRTI whose nasopharyngeal secretions were tested for HCoVs which infected with HCoV-229E (3.54%) and HCoV-OC43 (0.88%) and one sample was tested copositive for these viruses. 38 In line with the above findings, analysis of HCoV strains co-infectivity revealed that two (7.14%) out of the total number of our studied cases were co-infected with viral

ACKNOWLEDGMENTS
We would like to thank all the patients who kindly participate in our study. Also, the authors thank the entire staff of the National Influenza Center, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.